Skip to main content
. Author manuscript; available in PMC: 2013 Aug 15.
Published in final edited form as: Free Radic Biol Med. 2012 Jun 15;53(4):951–961. doi: 10.1016/j.freeradbiomed.2012.06.003

TABLE 3.

Differential Effects of Iron Chelator Pretreatment and Presence during Exposure on •NO2-Induced Membrane Tyrosine Nitration

Pretreatment Chelator Exposure Aqueous Phase 3-Nitrotyrosine
DTPA PO4 + + + +
DTPA 50 μM DFO —
DFO PO4 + + + +
DFO 400 μM DTPA + + + +

Red cell membranes (RCM), adhered to glass petri dishes, were covered with an aqueous solution containing either 400 μM DTPA or 50 μM DFO. After two 10 min treatments with periodic mixing (gas phase = air), systems were thoroughly rinsed to remove bound Fe complexes and remaining unbound chelator. RCM were then covered with PO4 buffer alone or buffer + chelator and exposed to 4.5 ppm •NO2 in air for 30 min with cyclic tilting (2 min/side). Following exposure, RCM were washed, membrane proteins isolated, and the presence of 3-nitrotyrosine residues determined by Western analysis. As described in Methods, the extent of 3-NT formation is denoted as extensive (++++), significant (+++), modest (++), little (+), or none (-).